A complete understanding of enzyme action requires a detailed knowledge about structure, dynamics and function. Although protein motions are the basis for enzyme function, this is the part of enzymology which is understood the least. Classically, enzyme reactions are studied by detecting substrate turnover. Our long-term goal is to examine enzyme catalysis in a nonclassical way, by characterizing motions in the enzyme during substrate turnover using dynamic NMR spectroscopy. This grant is focused on three reversible enzymes as model systems: the prolyl cis/trans isomerases human cyclophilin A (CypA) and Pin1, and adenylate kinase (Adk) from E.coli and Aquifex aeolicus. The static structures of these proteins are known. First, the intrinsic protein dynamics of the isomerases during catalysis will be characterized and compared to kinetic data measured for substrate interconversion. While it has been long hypothesized that motions are crucial for enzyme activity, the experimental detection of motions during catalysis will directly address questions, such as how the flexibility of the active site is linked to catalysis and what role collective motions play in enzyme function. Second, the interaction between CypA and the HIV-1 capsid protein will be explored mechanistically. This interaction is essential for HIV-1 replication, however, the role of CypA is not known. Using NMR and mutagenesis, structural rearrangements and the dynamic behavior of this enzyme/substrate complex will be studied. Finally, comparative studies on a hyper-thermophilic and a mesophilic enzyme pair will be performed as a second approach to examine the relation between protein dynamics and catalysis. This system provides the unique opportunity to use temperature as a powerful parameter to tune catalytic power and dynamics. Nuclear spin relaxation, NMR line-shape analysis, and NMR exchange spectroscopy are used as primary methods because they allow a residue-specific quantification of protein flexibility. Structure-based drug design is currently primarily based on static structures. However, many inhibitors bind to their targets in a way that can only be accomplished by conformational dynamics of the protein. The novel approach outlined in this proposal will lead to a deeper and more general understanding of protein dynamics, which should advance (enhance) the success of rational, structure based approaches for drug design.